PROJECT SUMMARY One of the most rapid ways that cells can alter gene expression is to control the processes of protein synthesis. By binding regulatory proteins to messenger RNAs, the cell can control protein production and quickly adapt to changes in environment without waiting for genes to be transcribed into mRNAs. The long-term goal of our laboratory is to characterize the protein/RNA interactions that post-transcriptionally regulate gene expression. The overall objective of this R15 application is to characterize the molecular mechanisms by which the RNA- binding protein LARP6 binds to RNA targets. The central hypothesis is that individual domains and motifs within LARP6 interact to coordinate specific RNA binding activitiy. The rationale driving this proposal is that the homologs of LARP6 from fish demonstrate robust structural stability and biochemical activity, enabling detailed in vitro studies of the molecular mechanisms of RNA/protein interactions. In particular, this work will expand the scope of mechanisms used by RNA recognition motif domains to interact with RNAs and thereby control gene expression. This hypothesis will be tested through three specific aims: (1) characterize the interdomain interactions within vertebrate LARP6; (2) determine the roles of highly conserved sequences within the LARP6 C-terminal domain; and (3) identify novel regulatory targets of vertebrate LARP6. In the first aim, the structures of subdomains of recombinant LARP6 proteins from bony fish will be determined to identify how the uncharacterized N-terminal domain interacts with the RNA binding domain. These results will show whether the RNA binding activity is modulated by specific intramolecular contacts. The second aim will test how the structure of the intrinsically disordered C-terminus responds to post-translational modification, and whether those changes affect RNA binding activity of the full-length protein. Quantitative in vitro assays with recombinant LARP6 domain constructs will measure molecular hydrodynamic radius, secondary structure content, and binding activity. The third aim will combine highly pure recombinant proteins, cellular RNAs, and cutting-edge sequencing technologies to identify novel cellular RNA ligands for vertebrate LARP6. The core innovation of this approach is in the use of a non-mammalian vertebrate system (fish) to study the biochemistry, structure, and function of a eukaryotic RNA-binding protein. As closer evolutionary relatives to mammals, telosts retain considerable genetic conservation so as to readily apply discoveries to human systems while also being sufficiently divergent to allow comparative analysis between species. This work is significant because it will identify new mechanisms of LARP6 structure and function, ultimately identifying novel RNA ligand sequences. These results will yield a broader understanding of how LARP6 exerts post-transcriptional control of gene expression, and enable direct tests of these mechanisms in physiological and genetic studies in the whole organism.